Immunoassay method for pro-gastrin-releasing peptide

ABSTRACT

To provide a more convenient and more accurate method of assaying ProGRP by improving the stability of ProGRP which is known to be unstable in a biological sample. 
     By using a blood sample in a condition in which a blood coagulation factor is not activated is used as a sample, the degradation of ProGRP is suppressed, whereby it is possible to store a sample for a long period of time and to improve the accuracy of an assay.

CROSS-REFERENCE

This application is the National Stage of International Application No.PCT/JP2007/073854, filed on Dec. 11, 2006, the contents of which arehereby incorporated by reference as if fully set forth herein.

TECHNICAL FIELD

The present invention relates to an assay method in which plasma is usedas a sample and the sample stability is remarkably improved in a systemfor detecting ProGRP (pro-gastrin-releasing peptide) by an immunoassay.

BACKGROUND ART

The usefulness of the relationship between lung cancer and theconcentration of blood gastrin-releasing peptide (GRP) has been known asreported by also Yamaguchi et al. (see Patent document 1). However, GRPis a physiologically active substance and loses its activity in anextremely short period of time in serum or plasma. Therefore, it wasdifficult to practically use it as an immunoassay. Yamaguchi et al.found that the antigen stability sufficient to withstand the practicaluse as an immunoassay can be achieved by specifically assaying, amongthree types of pro-gastrin-releasing peptides (ProGRPs) which areprecursors of GRP, an amino acid sequence of residues 31-98 (ProGRP31-98) which does not contain a physiologically active region (an aminoacid sequence of residues 1-27) and is a region common to the threetypes, and found that it is useful for diagnosis of lung cancer (seePatent document 1). According to this method, ProGRP can stably exist inserum or plasma equally up to 6 hours, therefore, it became possible toput it to practical use as an immunoassay method. Further, because anadvantage of the use of plasma is not particularly obtained, serum whichis generally used for assaying a cancer marker has come to be used as asample.

Patent Document 1

JP-A-6-98794

DISCLOSURE OF THE INVENTION

Problems that the Invention is to Solve

However, the stability of ProGRP 31-98 is still inferior to a generalantigen to be used in an immunoassay. An acceptable degree of decreasein the activity of ProGRP 31-98 in a sample seems to vary depending onthe manufacturer thereof. However, in the case of Immucheck F-ProGRPavailable from Sysmex Corporation, a sample is allowed to be storedunder refrigeration for at most 3 hours, in the case of a Serumlabo(registered trademark) ProGRP assay kit available from Fujirebio Inc., asample is allowed to be stored under refrigeration for at most about 24hours (either of them is a diagnostic agent which has been alreadyapproved by the Ministry of Health, Labour and Welfare), and bothproducts do not allow the storage at room temperature.

Further, it is construed that the stabilities thereof in plasma andserum are equal, therefore, only an assay using serum has been put inpractical use. In the case where a serum sample is stored for 3 hours tomore than 24 hours, it is necessary to freeze the sample. Therefore,labors of thawing the sample before use, removing a deposited mattergenerated by freezing and thawing by centrifugation before the assay arerequired, and so on, such insufficient sample stability significantlylowered the working efficiency. Further, many of the antigens includingtypical cancer markers such as CEA, AFP and CA 19-9, which areimmunologically assayed, can stably exist up to 7 days in refrigerationstorage. Therefore, only in the case where this assay is carried out,special operation and storage place are required, and so on, it has alsobecome a cause that lowers the efficiency of the test as a whole.

It has been construed that the reason why the sample stability of ProGRP31-98 is low is because the molecular weight thereof is low. Further,because a significant difference was not observed between the assayvalues of ProGRP 31-98 in serum and plasma in the range up to 6 hours,in which only a slight decrease in the activity thereof was observedeven in serum, it was not considered that there was a difference instabilities between serum and plasma.

Means for Solving the Problems

ProGRP is a protein having a molecular weight of 8000 to 10000, and adomain of an amino acid sequence of residues 31-98 with which an assayis carried out in an immunoassay of ProGRP 31-98 has a molecular weightof about 7800. It is known that the sample stability of a peptidemolecule having a low molecular weight is low as a general fact, and itcan be understood that a study of examining the cause of the low samplestability of ProGRP has not been reported. However, even in the casewhere a molecular weight is low like insulin (molecular weight: 5800) orthe like to be used as an immunodiagnostic agent, a peptide moleculethat can stably exist in serum or plasma for more than 5 days inrefrigeration storage exists, therefore, the inventor thought that theremust be some kind of mechanism of sample instability, which is unique toProGRP, in addition to its low molecular weight.

The inventors thought that because the ProGRP molecule per se can stablyexist in refrigeration storage for more than about 1 week, it is notonly attributable to ProGRP per se that makes it unstable in a sample,and there may be a substance which is present in blood and makes itunstable. Further, the inventor thought that because a GRP portion whichis a physiologically active region (an amino acid sequence of residues 1to 27) is not already contained in ProGRP 31-98, there may be apossibility that some kind of reaction other than a reaction occurringin vivo causes the sample instability unique to ProGRP.

As described in a standard book, when blood comes out of the body, morethan 10 substances called blood coagulation factors and fibrinolyticfactors are activated, and these respective substances inducedegradation or activation of precursor molecules of blood coagulationfactors, and in the end, induce activation of thrombin generated by thedegradation of prothrombin and formation of fibrin generated by thedegradation reaction of fibrinogen by thrombin thereby to coagulateblood, which is a well-known fact. A liquid component obtained byremoving a portion coagulated at this time results in serum. Most of theactivators of such blood coagulation factors are proteases includingthrombin and exist in serum. In plasma, the activation of thrombin isnot caused, and further, the activation of several blood coagulationfactors is not caused, although it depends on the anticoagulant. Thatis, in serum, activators and degraders of blood coagulation factors andfibrinolytic factors exist at a higher level in serum than in vivo or inplasma. Thus, the inventors presumed that there may be a relationshipbetween such a substance that exists in serum and does not exist orexists only at a low level in vivo and the instability of ProGRP 31-98.

In accordance with this presumption, the inventor examined a decrease inthe activity of thrombin, which is an activator of a blood coagulationfactor, by adding it to a ProGRP solution, and observed a significantdecrease in the activity of ProGRP in the solution with the addition ofthrombin. Accordingly, it was found that thrombin, which is an activatorof a blood coagulation factor, is one of the causes of the decrease inthe activity of ProGRP.

The inventor examined the stability of ProGRP by using plasma as asample with the use of a property that activators of blood coagulationfactors exist at a high level in serum and do not exist or exist only ata low level in vivo or in plasma, and succeeded in remarkably improvingthe sample stability of ProGRP, thus the present invention has beencompleted. It is possible to collect plasma from blood in a similarmanner to serum, and plasma can be collected from blood in asubstantially similar manner to serum, and can be used in an immunoassaywithout any problems. Further, by adding an inhibitor or an inactivatorof such an activator of a blood coagulation factor or a fibrinolyticfactor to a serum sample, a similar effect can be obtained.

Constitution of the Invention

The present invention provides, based on the above-mentioned findings, amethod of assaying ProGRP with which a more convenient and more accurateassay than a conventional method can be achieved by using a blood samplein a condition in which a blood coagulation factor is not activated,whereby fibrin formation is not caused and blood coagulation does notoccur.

The phrase, “a blood sample in a condition in which one or more bloodcoagulation factors are not activated” means a blood sample in acondition in which an agent that suppresses or lowers the activation ofany blood coagulation factor is added or a blood coagulation factor isremoved. As the agent that suppresses or lowers the activation of ablood coagulation factor, an agent such as EDTA, heparin, or citric acidcan be used, however, it is not limited to these.

Accordingly, as the blood sample in a condition in which bloodcoagulation does not occur, it is typical to use plasma. As the plasmadescribed here, any plasma sample to be used in a test can be used. Thatis, typical examples thereof include EDTA plasma, heparin plasma andcitric acid plasma, and also include other plasmas. Further, as for asalt to be used for each substance, an arbitrary salt can be selected.As for EDTA, typical examples thereof include 2K, 3K and the like. Asfor heparin, typical examples thereof include a sodium salt, a lithiumsalt and the like. However, other salts can also be selected.

Further, it is also possible to obtain an effect by adding a substancethat inactivates a blood coagulation factor to serum after collectingthe serum, removing a blood coagulation factor, or lowering the activitythereof.

It is proper to employ the present invention in an immunoassay method,however, the present invention can also be employed in any assay systemfor performing a ProGRP assay.

Advantage of the Invention

According to the present invention, a sample can be stably stored for along period of time. The storage period thereof in refrigeration storagecan be prolonged from 3 to 24 hours in a conventional method to about 1week. Further, it is also possible to store a sample at roomtemperature.

According to the present invention, it is not necessary to cryopreservea sample even in the case where the storage time of the sample exceeds 3to 24 hours. Therefore, labors of thawing the sample before use when thesample is frozen, removing a deposited matter generated by freezing andthawing by centrifugation are not required, and so on, the workingefficiency can be significantly improved. Further, it becomes possibleto store a sample for 1 week for which many of the other antigens to beimmunologically assayed can be stably stored in refrigeration storage,therefore, a special storage operation and storage place for a samplefor assaying ProGRP are not required.

Further, the present invention improves the long-term stability of asample in cryopreservation, and moreover, it improves the stabilitythereof at room temperature, therefore, labors of performing serumseparation or storage in refrigeration conditions can be omitted.Further, in the present invention, a decrease in the activity during theoperation at room temperature which cannot be avoided during the processof operation from blood collection to completion of assaying can besuppressed. Therefore, it becomes possible to assay ProGRP values moreaccurately.

It is considered that an instability degree of ProGRP in serum variesamong patients as a matter of course. Accordingly, it is conceivablethat in the case of a conventional method using serum as a sample, bythe decrease in the activity of ProGRP antigen during the process ofserum separation or storage, the serum exhibits a negative value uponassaying although it essentially has ProGRP corresponding to a positivevalue. It is considered that such occurrence of false negative testresults for the antigen activity can be prevented by employing thepresent invention, and thereby the detection sensitivity of small celllung cancer can be improved.

Incidentally, as the method of assaying ProGRP, there are a number ofdocuments, however, as an example in which plasma is used as a sample,there is only one report by Yamaguchi et al. However, this report onlyleads to the conclusion that serum and plasma are equal in terms of thesample stability, and it does not at all suggest the superior usefulnessof plasma in the present invention. Further, documents that describestability studies involved in other substances between serum and plasmaare shown below, however, it is obvious that they do not at all suggestthe assay method of the present invention.

Yamaguchi et al. (Japanese Patent No. 3210994) found a relationshipbetween ProGRP 31-98 and diagnosis of lung cancer and shows that aplasma sample can be used in the same manner as a serum sample up to astorage time of 6 hours. However, there is no description of thesuperiority of a plasma sample in terms of the storage stability. It isa work commonly performed in an immunoassay that the equivalence betweenplasma and serum as a sample subjected to an immunoassay is shown byusing both plasma and serum, and the constitution of the invention ofthis application that plasma has a superior usefulness is not inferableeasily from this document even by those skilled in the art.

Evans et al. (Clinical Biochemistry Vol. 34, pp. 107-112, 2001) examinedthe stability of a hormone having a physiological activity in serum andplasma and has reported that which of serum and plasma is better interms of the stability of the hormone varies depending on the type ofthe hormone. This shows that from the viewpoint of the stability of atest substance, a serum sample can be rather better than a plasmasample, which prevents those skilled in the art from arriving at thepresent invention of this application instead.

Boyanton et al. (Clinical Chemistry Vol. 48, pp. 2242-2247, 2002)studied the stabilities of 24 types of substances in serum and plasma.They showed that in either of the cases of serum and plasma, when serumseparation or plasma separation from blood cells was carried outimmediately after blood collection, the substances could be stably existfor 58 hours or more in either of serum and plasma. In addition, itconcluded that when separation was not carried out, the stability of thesubstances was better in serum. In this document, there is nodescription related to ProGRP and also it concluded that the stabilityis better in serum, therefore, we have no doubt that our invention hasnovelty.

Further, at present, three types of ProGRP diagnostic agents have beenapproved by the Ministry of Health, Labour and Welfare and are availableon the market and used in the clinical fields (medical coverage startedin 1996). However, there is no report that the stability of ProGRP 31-98in plasma is better than in serum. Therefore, it can be said that thisis clear evidence showing that the present invention has novelty.

The invention of this application that the stability of a sample isimproved by using a sample in a condition in which blood coagulationdoes not occur is a novel invention based on a mechanism which istotally different from any prior art, and exhibits an effect which couldnot be expected in the prior art.

EXAMPLES Example 1 Relationship Between Instability Of ProGRP Antigenand Thrombin

ProGRP 31-98 antigen (obtained from Abbott Laboratories in the U.S.A.)amino acid synthesized by the Fmoc method was added to a phosphatebuffer solution containing 1% bovine serum albumin and 2 mM calciumchloride. Thrombin (manufactured by Sigma-Aldrich Co.) was further addedthereto and after the mixture was stored at room temperature, theconcentration of ProGRP was assayed using an Architect ProGRP. The assayresults are shown in FIG. 1. From FIG. 1, because the degradation ofProGRP is observed in the presence of thrombin, it is suggested thatthrombin be related as a major cause of the degradation of ProGRP inserum. Thrombin is the activator of blood coagulation factor II and is asubstance which exists in serum at a high level. The details of theArchitect assay method are described in Example 3.

Example 2 Stabilization of ProGRP by Addition of PMSF(Phenylmethanesulfonyl Fluoride)

After PMSF (manufactured by Sigma-Aldrich Co.) was added to serum,ProGRP 31-98 antigen amino acid synthesized by the Fmoc method was addedthereto. After the mixture was stored at room temperature, theconcentration of ProGRP was assayed using the Architect ProGRP. Theassay results are shown in FIG. 2. From FIG. 2, it is found that adecrease in the activity of ProGRP is significantly suppressed in thepresence of PMSF. PMSF is a substance that is used to inactivate theactivity of an enzyme of a serine protease such as thrombin and thepresence of a protease such as thrombin can be considered to be thecause of ProGRP inactivation.

Example 3 Stabilization of ProGRP Antigen Using Plasma

ProGRP 31-98 antigen (obtained from Abbott Laboratories in the U.S.A.)synthesized by the Fmoc method was added to each of the serum and EDTAplasma obtained from the same blood donor and the mixtures were used asassay samples. After the assay samples were stored under refrigerationfor 1 day, 3 days or 7 days, or stored for 1 day at room temperature,the concentration of ProGRP was assayed. For the assay of theconcentration of ProGRP, a Serumlabo (registered trademark) ProGRP assaykit (Fujirebio Inc.) and the Architect ProGRP described below were used.The assay results are shown in FIG. 3 to FIG. 7.

Both the Serumlabo ProGRP and the Architect ProGRP are a method ofassaying the concentration of ProGRP 31-98 utilizing an immunoassay andthe assay results of both were equivalent. In both cases, when ProGRPantigen was allowed to exist in plasma, a significant improvement ofsample stability was observed compared with the case of serum. Thissignificant improvement of stability could be observed not only inrefrigeration storage but also in room temperature storage.

Although ProGRP can be stably stored for 1 day (until the following day)under the refrigeration storage condition in the method using serum, itcan be stably stored for 1 week or more in the case where plasma isused. The time required for a residual ratio to show 90% when a ProGRPresidual ratio is approximated as ProGRP residual ratio=e^(kt) (e:natural log, k: numerical constant, t: time) in accordance with anapproximation formula for an inactivation ratio of a general substanceshown by Evans et al. (Clinical Biochemistry, vol. 34, pp. 107-112,2001) is calculated to be 10.25 days under refrigeration when the assayvalue of a sample stored under refrigeration for 7 days using aSerumlabo (registered trademark) ProGRP assay kit is used. In a similarmanner, the time is calculated to be 1.26 days in the case of serum (theassay value of a serum sample stored under refrigeration for 1 day usinga Serumlabo (registered trademark) ProGRP assay kit is used for thecalculation). Thus, it is found that in the case where plasma is used,the stability thereof about 8 times as high as that of serum can beobtained.

In the currently available methods using serum, a ProGRP sample cannotbe stably stored at room temperature. In the case where plasma is used,it can be stably stored for 24 hours or more. The time required for aresidual ratio to show 90% when a ProGRP residual ratio is approximatedas ProGRP residual ratio=e^(kt) (e: natural log, k: numerical constant,t: time) is calculated to be 58 hours when the assay value of a samplestored at room temperature for 24 hours using Serumlabo is used, and itis possible to stably store a sample up to about 2 days even in roomtemperature storage.

Architect ProGRP Assay Method:

An anti-ProGRP 31-98 antibody (an antibody obtained in accordance withthe method described in Japanese Patent No. 3210994) was bound to acarboxyl group-modified magnetic microparticle (obtained from AbbottLaboratories in the U.S.A.) by a method using EDC(N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride(manufactured by Sigma-Aldrich Co.) and an antibody solid-phasedmicroparticle was prepared. Then, by adding the antibody solid-phasedmicroparticle to a Tris-HCl buffer solution containing Tween 20(manufactured by Kanto Chemical Co., Inc.), EDTA (ethylenediaminetetraacetic acid sodium salt) and sodium chloride, an antibodysolid-phased microparticle solution was prepared.

The anti-ProGRP 31-98 antibody (an antibody obtained in accordance withthe method described in Japanese Patent No. 3210994) was labeled with anacridinium derivative (obtained from Abbott Laboratories in the U.S.A.),and then by adding it to an MES buffer solution containing a surfactantand bovine serum albumin (Sigma-Aldrich Co.), a labeling solution wasprepared.

For an assay for the concentration of ProGRP, an Architect fullautomatic immunoassay analyzer (manufactured by Abbott Japan Co., Ltd.)was used. 50 μl of the antibody solid-phased microparticle solution wasmixed with 50 μl of a sample and a first reaction was started. Duringthe first reaction, ProGRP antigen binds to the antibody solid-phasedmagnetic particle and the amount bound corresponds to the concentrationof ProGRP in the sample. After 18 minutes, while being held by a magnet,the antibody solid-phased magnetic particle was washed with a phosphatebuffer solution dedicated to this device and 50 μl of the labelingsolution was further added thereto and the reaction was continued for anadditional 4 minutes. By this reaction, the labeled antibody binds tothe ProGRP on the magnetic particle. Because the amount of labeledantibody bound corresponds to the amount of ProGRP on the magneticparticle, if the concentration of ProGRP in the sample is low, a smallamount of labeled antibody results in binding to the magnetic particleand if the concentration of ProGRP in the sample is high, a large amountof labeled antibody results in binding to the magnetic particle.

Then, after washing is carried out with the phosphate buffer solutiondedicated to this device, a luminescence signal was observed using aluminescence pretrigger reagent and a trigger reagent dedicated to thisdevice. By using a solution with a known concentration preparedseparately as a standard solution, a standard curve was prepared by thefour parameter logistic method, and by calculating a signal obtainedfrom the sample into the concentration of ProGRP, the concentration ofProGRP in the sample was determined.

Example 4 Comparison of a Variety Types of Plasmas

Recombinant ProGRP 31-98 obtained in accordance with the methoddescribed in Japanese Patent No. 3210994 was added to each of matchedsamples of EDTA plasma, lithium heparin plasma, citric acid plasma,sodium heparin plasma and serum obtained from the same blood donor andthe mixtures were stored at room temperature for 24 hours or storedunder refrigeration for 7 days and the concentration of ProGRP wasassayed in the same manner as described in Example 3. From the assayvalue of the sample left at room temperature for 24 hours, the assayvalue of 0 hour was subtracted, and the resulting value was taken as aProGRP residual ratio after 24-hour room temperature storage. From theassay value of the sample stored under refrigeration for 7 days, theassay value of 0 hour was subtracted, and the resulting value was takenas a ProGRP residual ratio after 7-day refrigeration storage.

In FIG. 7, the ProGRP residual ratio in each plasma after 7-dayrefrigeration storage is shown. In FIG. 8, the ProGRP residual ratio ineach plasma after 24-hour room temperature storage. From FIGS. 7 and 8,the ProGRP residual ratios are significantly higher in any of theplasmas compared with in serum and, in other words, it is obvious thatthe sample stability is significantly improved in any of the plasmas. Inaddition, because the residual ratios are almost 100% in 7-dayrefrigeration storage in plasma, it is obvious that almost total amountof ProGRP is stably stored in plasma up to 7 days under refrigeration.

Example 5 Time Course

ProGRP 31-98 antigen amino acid synthesized by the Fmoc method was addedto each of matched samples of EDTA plasma and serum obtained from thesame blood donor and the mixtures were stored at room temperature orunder refrigeration and the concentration of ProGRP was assayed by themethod described in Example 3. The values up to 24 hours are shown bythe average of 3 donors and the values up to 72 hours are shown by theaverage of 2 donors. The ProGRP residual ratios in refrigeration storageand the ProGRP residual ratios in room temperature storage are shown inFIG. 9 and FIG. 10, respectively. The storage times shown in thedrawings are 0, 1, 3, 6, 7, 9 and 24 hours (both refrigeration storageand room temperature storage until this time) and 72 hours (onlyrefrigeration storage).

From FIG. 9, as Yamaguchi et al. shows, in refrigeration storage, serumand plasma have an equivalent ProGRP stability in storage up to 6 hours.However, the difference gradually becomes significant after 6 hours, andwhen a ProGRP sample is stored for 24 hours or more, plasma apparentlycan show better stability than serum.

From FIG. 10, in room temperature storage, a significant difference inthe stability of ProGRP between serum and plasma has already occurred atthe time that exceeds 3 hours. In addition, when a sample is stored for6 hours or more, it is obvious that plasma can show apparently betterstorage stability than serum. In the case where ProGRP is stored inserum, the activity of ProGRP decreases to about 80% at the time of6-hour storage. When a sample is stored for the purpose of a diagnosticagent, generally a residual activity up to 90% (a decrease in theactivity by 10%) is the acceptable limit. Because in the case where asample is stored at room temperature in serum, the activity of ProGRPdecreases to about 80% at the time of 6-hour storage, it can be saidthat the result shown in FIG. 10 is consistent with the descriptionmatter in the Serumlabo (registered trademark) ProGRP assay kit and theImmucheck F-ProGRP kit of the approved products which do not allow roomtemperature storage. On the other hand, in the case where ProGRP isstored in plasma, the residual ratio of activity is about 90% even inthe case where it is stored for 24 hours even in room temperaturestorage, therefore, it is considered that stable storage up to about 24hours can be achieved. Yamaguchi et al. shows that plasma can be usedwithin the range that allows ProGRP to exist stably in serum, howeverthe present invention is different from the invention of Yamaguchi etal. in the point that the storage stability of ProGRP in a blood samplethat is sufficient to be used for the purpose of a diagnostic agent canbe secured even in a storage condition or storage time in which ProGRPcannot stably exist in serum and it is considered that the usefulness ofthe present invention can be shown.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows ProGRP residual ratios in room temperature storage in thepresence or absence of thrombin or in serum.

-   -   ProGRP residual ratio    -   Storage time (hours) in room temperature storage    -   ♦: Solution without addition of thrombin    -   □: Solution with addition of thrombin    -   X: Serum

FIG. 2 shows ProGRP residual ratios in room temperature storage in serumwith or without addition of PMSF.

-   -   ProGRP residual ratio after sample was left    -   Leaving time (hours) in room temperature storage    -   ♦: Serum (without addition of PMSF)    -   ▪: Serum with addition of PMSF

FIG. 3 shows ProGRP residual ratios in refrigeration storage in serum orplasma.

-   -   Assayed by Serumlabo ProGRP method    -   ProGRP residual ratio after sample was left    -   Leaving time (days) in refrigeration storage    -   ♦: Serum    -   ▪: Plasma

FIG. 4 shows ProGRP residual ratios in room temperature storage in serumor plasma.

-   -   Assayed by Serumlabo ProGRP method    -   ProGRP residual ratio after sample was left    -   Leaving time (hours) in room temperature storage    -   ♦: Serum    -   570 : Plasma

FIG. 5 shows ProGRP residual ratios in refrigeration storage in serum orplasma.

-   -   Assayed by Architect ProGRP method    -   ProGRP residual ratio after sample was left    -   Leaving time (days) in refrigeration storage    -   ♦: Serum    -   ▪: Plasma

FIG. 6 shows ProGRP residual ratios in room temperature storage in serumor plasma.

-   -   Assayed by Architect ProGRP method    -   ProGRP residual ratio after sample was left    -   Leaving time (hours) in room temperature storage    -   ♦: Serum    -   ▪: Plasma

FIG. 7 shows ProGRP residual ratios after 7-day refrigeration storage inserum or plasma.

-   -   ProGRP residual ratio after 7-day refrigeration storage    -   Serum    -   EDTA plasma    -   Lithium heparin plasma    -   Citric acid plasma    -   Sodium heparin plasma        -   : Donor

FIG. 8 shows ProGRP residual ratios after 24-hour room temperaturestorage in serum or plasma.

-   -   ProGRP residual ratio after 24-hour room temperature storage    -   Serum    -   EDTA plasma    -   Lithium heparin plasma    -   Citric acid plasma    -   Sodium heparin plasma        -   : Donor

FIG. 9 shows ProGRP residual ratios in refrigeration storage in serum orplasma.

-   -   ProGRP residual ratio    -   Storage time (hours) in refrigeration storage    -   ♦: Serum    -   ▪: EDTA plasma

FIG. 10 shows ProGRP residual ratios in room temperature storage inserum or plasma.

-   -   ProGRP residual ratio    -   Storage time (hours) in room temperature storage    -   ♦: Serum    -   ▪: EDTA plasma

1. A method of assaying ProGRP in a blood sample comprising the stepsof: a)contacting said blood sample with an antibody against ProGRP for atime and under conditions sufficient for formation of antibody/ProGRPcomplexes, wherein said blood sample comprises one or more unactivatedblood coagulation factors; and b) detecting presence of saidantibody/ProGRP complexes, presence of said complexes indicatingpresence of ProGRP in said blood sample.
 2. The method of assayingProGRP according to claim 1, wherein a said blood sample comprisesunactivated thrombin.
 3. The method of assaying ProGRP according toclaim 1 or claim 2, wherein plasma is used as the sample.
 4. (canceled)5. The method of assaying ProGRP according to claim 1, wherein theregion covering amino acids 31-98 of ProGRP is assayed.
 6. The method ofassaying ProGRP according to claim 1, wherein said sample is stored formore than 6 hours in refrigeration storage after collection.
 7. Themethod of assaying ProGRP according to claim 6, wherein said sample isstored for more than 24 hours in refrigeration storage after collection.8. The method of assaying ProGRP according to claim 1, wherein saidsample is left or stored at room temperature after collection.
 9. Amethod of improving the in vivo stability of a serum sample containingProGRP comprising the step of adding to a serum sample containing ProGRPobtained from a subject a stabilizing amount of at least one serumprotease inhibitor.
 10. The method of claim 9, wherein the serineprotease inhibitor is phenylmethylsulfonyl fluoride.
 11. The method ofclaim 9, wherein the at least one serum protease inhibitor is added to asample collection vessel after serum is added to the sample collectionvessel.
 12. A ProGRP-stabilizing composition, wherein said compositioncomprises at least one serine protease inhibitor.
 13. A samplecollection vessel containing the composition of claim
 12. 14. A kit forstabilizing ProGRP in a serum sample, the kit comprising: (a) at leastone serum protease inhibitor; and (b) instructions for using the kit.15. The kit of claim 14 wherein the at least one serum proteaseinhibitor is phenylmethylsulfonyl fluoride.